The recent growth in many areas of biotechnology has increased the demand to perform a variety of studies, commonly referred to as assays, of biochemical systems. These assays include for example, biochemical reaction kinetics, DNA melting point determinations, DNA spectral shifts, DNA and protein concentration measurements, excitation/emission of fluorescent probes, enzyme activities, enzyme co-factor assays, homogeneous assays, drug metabolite assays, drug concentration assays, dispensing confirmation, volume confirmation, solvent concentration, and solvation concentration. Also, there are a number of assays which use intact living cells and which require visual examination.
Assays of biochemical systems are carried out on a large scale in both industry and academia, so it is desirable to have an apparatus that allows these assays to be performed in convenient and inexpensive fashion. Because they are relatively easy to handle, are low in cost, and generally disposable after a single use, multiwell plates are often used for such studies. Multiwell plates typically are formed from a polymeric material and consist of an ordered array of individual wells. Each well includes sidewalls and a bottom so that an aliquot of sample may be placed within each well. The wells may be arranged in a matrix of mutually perpendicular rows and columns. Common sizes for multiwell plates include matrices having dimensions of 8×12 (96 wells), 16×24 (384 wells), and 32×48 (1536 wells).
Typically, the materials used to construct a multiwell plate are selected based on the samples to be assayed and the analytical techniques to be used. For example, the materials of which the multiwell plate is made should be chemically inert to the components of the sample or any biological or chemical coating that has been applied to the plate. Further, the materials should be impervious to radiation or heating conditions to which the multiwell plate is exposed during the course of an experiment and should possess a sufficient rigidity for the application at hand.
In many applications, a transparent window in the bottom of each sample well is needed. Transparent bottoms are primarily used in assay techniques that rely on emission of light from a sample and subsequent spectroscopic measurements. Examples of such techniques include liquid scintillation counting, techniques which measure light emitted by luminescent labels, such as bioluminescent or chemoluminescent labels, fluorescent labels, or absorbance levels. Optically transparent bottom wells also lend the advantage of microscopic viewing of specimens and living cells within the well.
Currently, optically transparent and UV transparent bottomed multiwell plates exist in the market and are used for the purposes described. These plates typically are a hybrid of different polymeric materials, one material making up the well walls and another making up the bottom portion of the wells.
Ideally, plates to be used for spectroscopic and microscopic measurement would have well bottoms made from glass. Glass has the advantage of being chemically inert, has superior optical properties in the visible range, is rigid, and is highly resistant to any deformation process caused by heating, due to its high melting temperature. Further and unlike most polymers, glass can be formulated and processed to provide a surface which produces very little background signal and which may be manufactured to extreme smoothness. Still, its surface may be easily coated or otherwise altered in order to promote attachment of specific targeted molecules. For example, a silane coating may be applied to the glass in order to extend any variety of functional groups such as amine functionalities, for example. Such amine functionality may can be effectively used to immobilize reactive molecules of the types commonly used in biological assays and testing procedures, e.g., to immobilize specific binding members (e.g., antigens, ligands, and haptens), entire cells, proteins (e.g., binding proteins, receptor proteins, antibodies and antibody fragments), nucleic acids (e.g., RNA and DNA molecules), tissue and the like. Further, the use of a polylysine coating on glass cover slips to grow nerve cells is a standard procedure.
Unfortunately, while it is simple to make glass in sheets, it is not possible to injection mold articles made from glass, and it is extremely difficult to press a molten gob of glass into an industry standard assay plate format. One solution to the problem, offered by the present invention, is to combine an injection molded polymeric upper plate molded to form the wells of a microplate, with a substantially flat transparent glass lower plate to form the well bottoms. In order to accomplish this result, the inventors considered several known methods for combining glass and plastic. Two commonly employed methods of joining these types of materials are by means of adhesive bonding and by means of insert molding.
The use of adhesives to bond together the material forming the well bottoms and material forming the well walls is expensive and leads to contamination of the biologically sensitive well surface. Low molecular weight species from the polymeric material making up the sidewalls of the wells, as well as species within the adhesive itself, tend to migrate through the adhesive and onto the transparent bottom surface. When this occurs, biomolecules can not properly react with the surface as intended under particular assay conditions. Adhesives which are UV cured or UV stabilized also have the tendency to absorb UV light, which may result in altering fluorescent readings taken from a detector located above or below the plate. The effect of the UV light is to non-specifically modify the signal by non-specific fluorescence thus creating undesired background readings which are highly variable from well to well and from plate to plate.
Insert molding is another common technique for joining together polymeric and glass parts. In this manufacturing method, the polymer portion is molded against or around the glass portion. Since the polymer has a much lower melting temperature than the glass, the glass remains in solid form while the liquid polymer is pressed against it. Once hardened, the polymer/glass interface remains attached only by weak interactions. One way of increasing the mechanical strength of the connection is to mold around or encapsulate the glass with the polymer, e.g. in glass bottomed ashtrays. Unfortunately, using this technique to combine polymeric parts with glass sheets of microscope coverslip thickness, as described in multiwell plate manufacture, is not practical because the mechanical strength of glass at such thinness is extremely low.